Name: selective capture of 5-hydroxymethylcytosine from genomic dna
Uploaded: 2012-10-05T13:00:00
Description: Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Denne undersøgelse blev støttet delvist af National Institutes of Health (GM071440 til CH og NS051630/MH076090/MH078972 til PJ).

1. Genomisk DNA Fragmentation Fragment genomisk DNA ved anvendelse af lydbehandling til et ønsket størrelsesområde egnet til genomet hele sekventering platform. (Vi normalt lydbehandling for at ~ 300 bp.) Kontroller størrelsesfordelingen af den fragmenterede genomiske DNA på 1% agarosegel (figur 1). 2. DNA-forberedelse Bestemme de udgangsmaterialer DNA beløb baseret på forekomsten af ​​5-HMC i genomisk DNA. Sid...

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5-hydroxymethylcytosine (5-HMC) er en nylig identificeret epigenetisk modifikation til stede i væsentlige mængder i visse mammale celletyper. Metoden, der præsenteres her, er til bestemmelse af genom-dækkende udbredelse af 5-HMC. Vi anvender T4 bakteriofag β-glucosyltransferase at overføre en manipuleret glucosedel indeholder en azidgruppe på hydroxylgruppen i 5-HMC. Af azidgruppen kan modificeres kemisk med biotin til påvisning, affinitetsberigelse, og sekventering af 5-HMC-holdige DNA-fragmenter i mammale geno...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Katalog # </td><td> Kommentar </td></tr><tr><td></td><td></td><td></td><td> Reagenser </td></tr><tr><td> 5M Natriumchlorid (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> 0,5 M pH 8,0 Ethylendiamintetraeddikesyre (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1 M Trizma-base (Tris) pH 7,5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1 M, pH 7,4 </td><td> Invitrogen </td><td> 15.630 </td><td></td></tr><tr><td> Magnesiumchlorid (MgCl2) 1M </td><td> Ambion </td><td> AM9530g </td><td></td></tr><tr><td> Dimethylsulfoxid (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Tween 20 </td><td> Fisher bioreagenser </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-biotinkonjugat </td><td> Klik Kemi Tools </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-dithiothreitol, ultrarent (DTT) Superpure </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> QIAquick Nucleotide Removal Kit </td><td> Qiagen </td><td> 28.304 </td><td></td></tr><tr><td> Micro Bio-Spin 6 Column </td><td> Bio-Rad </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Streptavidin C1 </td><td></td><td></td><td></td></tr><tr><td> Qiagen MinElute PCR Purification Kit </td><td> Qiagen </td><td> 28.004 </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N3-glucose </td><td> Active Motif </td><td> 55.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Enzym </td></tr><tr><td> β-glucosyltransferase (β-GT) </td><td> New England Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> Udstyr </td></tr><tr><td> Sonication anordning </td><td> Covaris </td><td></td><td></td></tr><tr><td> Desktop centrifuge </td><td></td><td></td><td></td></tr><tr><td> Vandbad </td><td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Gel kører apparater </td><td> Bio-Rad </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Labquake Tube Shaker </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tube Shaker </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> Magnetic Separation Stand </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> Qubit 2,0 Fluorometer </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> Reagens setup 10 X β-GT Reaction Buffer (500 mM HEPES pH 7,9, 250 mM MgCl2) 2 X binding og vask (B &amp; W) puffer (10 mM Tris pH 7,5, 1 mM EDTA, 2 M NaCI, 0,02% Tween 20) . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-methylcytosin (5-Mc) udgør ~ 2-8% af de samlede cytosiner i human genomisk DNA og påvirker en lang række biologiske funktioner, herunder genekspression, vedligeholdelse af genom integritet, parentale prægning, X-kromosom inaktivering, regulering af udvikling, aldring og kræft 1. For nylig blev tilstedeværelsen af en oxideret 5-mC, 5-hydroxymethylcytosine (5-HMC), fundet i mammale celler, især i embryostamceller (ES) celler og neuronale celler 2-4. 5-HMC genereres ved oxidationen af 5-mC katalyseret af TET familien jern (II) / α-ketoglutarat-afhængig dioxygenases 2, 3. 5-HMC foreslås at være involveret i vedligeholdelse af embryonale stamceller (MES) celle, normal hæmatopoiese og maligniteter, og zygote udvikling 2, 5-10. For bedre at forstå funktionen af ​​5-HMC, en pålidelig og enkel sekventering er afgørende. Traditionel bisulfit sekventering kan ikke skelne 5-HMC fra 5-MC 11 </sup&gt;. At optrævle biologi 5-HMC, har vi udviklet en meget effektiv og selektiv kemisk tilgang til at mærke og fange 5-HMC, at drage fordel af en bakteriofag enzym, der tilføjer en glucosedel til 5-HMC specifikt 12. Her beskriver vi en simpel totrinsprocedure til selektiv kemisk mærkning af 5-HMC. I det første mærkning trin 5-HMC i genomisk DNA mærket med en 6-azid-glucose katalyseret af β-GT, en glucosyltransferase fra T4-bakteriofag på en måde, som overfører 6-azid-glucose til 5-HMC fra modificeret cofaktor, UDP-6-N3-Glc (6-N3UDPG). I det andet trin, biotinylering, er en disulfid biotin-linker bundet til azidgruppen af ​​klik kemi. Begge trin er særdeles specifikke og effektive, hvilket fører til fuldstændig mærkning uanset forekomsten af ​​5-HMC i genomiske regioner og give yderst lav baggrund. Efter biotinylering af 5-HMC, de 5-HMC-holdige DNA-fragmenter derefter selektivt fangetved hjælp af streptavidin-perler i en densitet-uafhængig måde. De resulterende 5-HMC-berigede DNA-fragmenter kan anvendes til efterfølgende analyser, herunder næste generation sekventering. Vores selektiv mærkning og capture-protokol giver høj følsomhed, der gælder for enhver kilde til genomisk DNA med variable / diverse 5-HMC overflod. Selvom hovedformålet med denne protokol er dens downstream anvendelse (dvs.., Næste generation sekventering at kortlægge 5-HMC distribution i genomet), det er foreneligt med enkelt-molekyle, real-time SMRT (DNA) sekventering, der er kan levere single-base opløsning sekventering af 5-HMC.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Beskrives en totrins-mærkning ved anvendelse β-glucosyltransferase (β-GT) for at overføre et azid-glucose til 5-HMC, efterfulgt af klik kemi til at overføre en biotin linker til nem og densitet-uafhængig berigelse. Denne effektive og specifikke mærkning metode gør det muligt berigelse af 5-HMC med ekstremt lav baggrund og højkapacitetsidentifikation epigenomic kortlægning via næste generation sekventering.

Selektiv opsamling af 5-hydroxymethylcytosine fra genomisk DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

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